Phytochemical Study of Tanacetum Sonbolii Aerial Parts and the Antiprotozoal Activity of its Components

The genus Tanacetum includes some popular endemic species of the flora of Iran, with important medicinal properties. In a project, directed at structurally interesting bioactive metabolites from Iranian endemic species, we studied Tanacetum sonbolii Mozaff. Eight compounds comprising six phenolic and two terpenoidal compounds were isolated from the ethyl acetate extract of the aerial parts of the plant by normal and reverse phase chromatography. Their structures were established mainly by 1D and 2D NMR spectroscopic techniques, including 1H-1H COSY, HSQC and HMBC methods and confirmed by comparing their NMR data with those reported in the literature. The compounds namely: 2,4-dihydroxy-6-methoxyacetophenone (1), apigenin (2), 5-desmethylsinensetin (3), 5-desmethylnobiletin (4), 8-methoxycirsilineol (5), scopoletin (6), ursolic acid (7), and β-sitosterol (8). In-vitro antiprotozoal activity of compounds 1, 3, and 5 were evaluated against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum parasites and also toxicity against rat myoblast (L6) cells. Compound 5 showed promising activity against T. b. rhodesiense.


Introduction
Tanacetum is a remarkable genus of the Asteraceae family, comprises about 14 sections and 200 species, native to Europe, Mediterranean region, Western Asia and North America (1). Tanacetum species have been used for centuries as valuable herbal remedy in traditional medicine because of their cytotoxic, antimicrobial, antiviral and anti-inflammatory activities (2, 3). Tanacetum parenthium (feverfew) which is listed in European Pharmacopeia as a traditional remedy has been used for the treatment of migraine (4, 5). It has been reported that sesquiterpene lactones are the major classes of the secondary metabolites from the genus Tanacetum. Also, it has been reported that the flavonoids, coumarins, and triterpenoids are the main classes of compounds in this genus (6-8). In the flora of Iran, the genus Tanacetum consists of 36 annual and perennial species, 16 of which are endemic (9)M.V.Agab. & Wagenitz (Asteraceae, Anthemideae.
Tanacetum sonbolii Mozaff. is an endemic species of West Azerbaijan province of Iran.
A literature survey showed that there has been no phytochemical study on T. sonbolii, apart from an analysis of the essential oil (10). Antioxidant activity, protective effect against hydrogen peroxide-induced oxidative stress in K562 cell line, and effect on PTZ-induced seizures in male mice have been reported for various extracts prepared from different parts of the plant (10-12). As a part of our ongoing research on discovering new and potentially bioactive secondary metabolites from Iranian species (13-15), we investigated the phytochemical compostion of ethyl acetate extract from the aerial parts of T. sonbolii.
Here we report the isolation and structure elucidation of eight compounds by applying 1D and 2D NMR spectroscopy. Compounds 1, 3, and 5 were tested against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum parasites, and showed good activities against T. b. rhodesiense.

Experimental
General experimental procedures NMR spectra were mesured at 18 °C on a Bruker Avance III 500 MHz spectrometer, 500.13 MHz for 1 H, and 125.77 MHz for 13 C. A 1-mm TXI-microprobe with z-gradient was used for 1 H-detected experiments. 13 C NMR spectra were recorded with a 5 mm BBO probe head with z-gradient. The spectra were analyzed using Bruker TopSpin 3.1 software. Deuterated solvents for NMR (100% D) were purchased from Armar Chemicals. HPLC separations were performed on a Knauer HPLC system consisting of a mixing pump 1000 with degasser module, PDA detector, and an autosampler. Knauer Eurospher П 100-5 RP C18 column (5 μm, 4.6 × 250 mm) and SunFire Prep C18 ODB (5 μm, 19 × 50 mm i.d.) columns were used for reversed-phase analytical and semipreparative separations, respectively. The solvents used for extraction and column chromatography were of technical grade and were distilled before use (Emertat, Iran). The solvents used for HPLC were of HPLC grade (Merck, Germany). Silica gel (70-230 mesh) was used for column chromatography, and pre-coated silica gel F254 (20 × 20 cm) plates (Merck, Darmstadt, Germany) for TLC. Anisaldehye (Merck, Germany) was used for visualization of the TLC plates.

Plant material
Aerial parts of Tanacetum sonbolii were collected from Takab mountains, West-Azerbaijan province, Iran, in June 2015 and identified by Dr. Ali Sonboli. A voucher specimen (MPH-2556) has been deposited in the Herbarium of the Medicinal Plants and Drugs Research Institute of Shahid Beheshti University, Tehran, Iran.

Extraction and isolation
The air-dried aerial parts of T. sonbolii (1.6 kg) were crushed and extracted with ethyl acetate (5 × 7 L) by maceration at room temperature. The extract was concentrated in vacuum, to afford 60 g of a dark gummy residue. The residue was separated on a silica gel column (70-230 mesh, 6.0 × 120 cm, 850 g) with a gradient of n-hexane-EtOAc (100:0 → 0:100) as eluent, followed by increasing concentration of MeOH (up to 100%) in EtOAc. On the basis of TLC analysis (detection at 254 nm, and after spraying anisaldehyde-sulfuric acid reagent), fractions with similar compositions were pooled to yield 21 combined fractions.

Statistical analysis
All measurements were expressed as the mean ± standard deviations (SD) in triplicate manner. Excel 2010 was employed for analyzing data. The IC 50 values were calculated by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software. The selectivity index was calculated as IC 50 for L-6 cells/IC 50 for parasites.
In-vitro antiprotozoal activity of compounds 1, 3, and 5 were studied against T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum. The compounds showed good inhibition against T. b. rhodesiense. Compound 1 inhibited the growth of the parasite equal to 95.2% at the concentration of 10 µg/mL,  while Melarsoprol as the reference compound showed 100% inhibition. Also, compound 3 and 5 showed considerable inhibition equal to 99.6% and 96.8% at 10 µg/mL concentration, respectively. Accordingly, these compounds were considered for further study to evaluate the IC 50 values against T. b. rhodesiense and cytotoxicity against L6 cells. The results are presented in Table 1; Compound 5 exhibited the most inhibitory activity against T. b. rhodesiense with IC 50 value of 6.1 μM and selectivity index (SI) equal to 5.4.

Conclusion
In the present study, eight known compounds were isolated from the aerial parts of Tanacetum sonbolii. Their structures were elucidated by means of extensive 1D, 2D NMR spectroscopy. Also the in-vitro antiprotozoal activity of the compounds was studied. Compounds 1, 3, and 5 were the active constituents for growth inhibition of T. brucei rhodesiense at 10 µg/mL concentration and the most active compound was compound 5 with IC 50 value of 6.1 μM and selectivity index of 5.4.
Health Institute), and T. Hettich (Group of Prof. Dr. G. Schlotterbeck, School of Life Sciences, University of Applied Sciences, Institute of Chemistry and Bioanalytics, Muttenz) for the HRAPCIMS data, respectively. Special thanks go for assisting on NMR spectra measurement at university of Basel, division of pharmaceutical biology (Group of Prof. Dr. M. Hamburger).